Project Summary Bacterial vaginosis (BV) is the most prevalent polymicrobial condition in women of reproductive age, and is associated with a significantly higher incidence of new HIV-1 infections in women. BV is a microbial shift condition in which commensal lactobacilli, associated with a healthy vaginal microbiota, are displaced by an overgrowth of mixed pathogenic bacterial populations. When BV alters the homeostasis of the cervicovaginal mucosa, perturbation of the lactobacilli-dominated vaginal microbiota leads to an increase in pH, an increase in nonresident or underrepresented microbes, and the ascension of these microbes into the aseptic upper genital tract. The cervicovaginal mucosa possesses multiple mechanisms of innate host defense, the foremost of which is a protective epithelia that represents the first point of contact for invading pathogens. In the current application, we present evidence of a novel, BV-mediated mechanism that augments HIV-1 infection of the cervicovaginal mucosa. We reveal that BV-associated bacteria (BVAB) induce cervicovaginal epithelia to produce specific matrix metalloproteinases (MMPs), which disrupt the protective epithelial barrier likely by degrading tight junctions. In addition, cervicovaginal fluids (CVF) from BV patients decreased cervicovaginal epithelial barrier integrity; MMP concentration and activity were elevated in CVF from BV patients and in BVAB-induced cervicovaginal epithelia; and, conditioned media from BVAB-stimulated endocervical cells increased HIV-1 transmission through cervicovaginal epithelia. Our hypothesis is that BV induces MMPs that damage the cervicovaginal epithelial barrier, thus enhancing the ability of HIV-1 to infect underlying target cells. In Aim 1, we will evaluate the role of BV-induced MMPs in increasing HIV-1 infection in cervicovaginal tissues, and propose studies to answer the following questions: To what extent does BV CVF increase HIV-1 infection of organotypic cervicovaginal tissues? Does resolution of BV abrogate its membrane-damaging potential? Will the addition of MMPs to healthy CVF mimic the membrane-disrupting potential of BV CVF? Can MMP inhibitors added to BV CVF reduce HIV-1 infection of organotypic cervicovaginal tissues? We expect to identify key MMPs that act as barrier disrupters during BV, and to identify commercially available MMP inhibitors that block these pro-viral consequences of natural host defense against BV. In Aim 2, we will elucidate the mechanism by which BV activates MMPs to decrease barrier function in cervicovaginal epithelia, and propose studies to answer the following: Are natural tissue inhibitors of MMPs (TIMPs) down-regulated during BV, and can adding TIMPs to BV CVF reduce MMP activity? Which cervicovaginal barrier proteins are affected by BV- enhanced MMP activity, and can the addition of synthetic MMP inhibitors or TIMPs reduce their cleavage by BV CVF? Can increasing D-lactic acid levels reduce expression of EMMPRIN and activated MMPs in cervicovaginal tissues? Collectively, our studies will delineate the MMP-mediated dysregulation that occurs during BV, and identify targets for future development as preexposure prophylaxis for women.